Alzheimer?s is a devastating disease involving chronic, progressive neurodegenerative processes and decline in brain cognitive function that ultimately lead to death. Accumulation of aggregates of amyloid-beta and hyperphosphorylated tau protein in the brain causes synaptic dysfunction and loss of neurons, although the precise mechanisms underlying the disease remain unknown. There are few drugs available to treat Alzheimer?s disease, and they provide only limited benefits. In fact, the economic damages from Alzheimer?s disease reach hundreds of billion dollars every year in the US alone. Many candidate drugs have been evaluated in clinical trials in the last decade, but none has been approved. Thus, it is important to identify new drugs based on novel targets. The EphA4 receptor tyrosine kinase has recently emerged as a novel promising target for counteracting neurodegeneration and cognitive deficits in Alzheimer?s disease and other neurodegenerative diseases. EphA4 receptor signaling can promote neurotoxicity when aberrantly induced by amyloid-beta oligomers and ephrin ligands. The kinase domain of EphA4, which is responsible for the neurotoxic effects, is a druggable target. However, small molecule kinase inhibitors targeting EphA4 with selectivity and high affinity remain to be identified. Here we propose to perform two high-throughput screening campaigns of EphA4. One screen will deploy Protein Thermal Shift (PTS) as the assay format and the other screen will deploy a kinase activity assay format. The goal of these screens is to identify as broad a panel of modulators of the kinase-dependent functions of EphA4 as possible. Use of two formats should maximize our ability to identify a large repertoire of modulators with diverse modes of action and ultimately develop an inhibitory compound with high selectivity for EphA4. We anticipate that the PTS screen will identify compounds that stabilize the inactive conformation of the EphA4 intracellular region by binding not only to the kinase domain but also to regulatory regions outside this domain. The second screen will deploy a novel in vitro kinase assay configuration we have developed, where the inactive EphA4 intracellular region serves as the physiological substrate for the catalytically competent EphA4 intracellular region. We anticipate that this assay will identify not only compounds that target the EphA4 ATP binding pocket, but also allosteric inhibitors with novel mechanisms of action. The top hits identified in the two screens and their derivatives will be characterized and improved through rounds of secondary and tertiary biochemical and cell-based assays already established in the laboratories of the PIs and their collaborators, complemented by structure-guided approaches such as in silico docking and X-ray crystallography. These assays will provide formal hit validation, selectivity profiling and initial characterization of mechanisms of action and activities in neurons. We anticipate that this project will result in the identification of several novel EphA4 selective inhibitors that are suitable for advancement to future studies in animal models of Alzheimer?s and other neurodegenerative diseases.